Measurement and uses of oxidative status

ABSTRACT

The present invention provides a method of determining the overall oxidative status of a body fluid or a tissue of a patient by measuring the oxidation-reduction potential (ORP) of the body fluid or tissue. The method has been found to be useful in the diagnosis, evaluation and monitoring of patients who have suffered a trauma (such as a head injury), patients suspected of being critically-ill, patients who have a viral infection, and patients suspected of having a myocardial infarction. The method has also been found useful in monitoring and evaluating exercise performance in patients. In addition, the method has been found useful in monitoring and evaluating stored blood products and patients who will receive such a product.

This application is a continuation of U.S. application Ser. No.12/121,945, filed May 16, 2008, which claims the benefit of U.S.Provisional Application No. 60/938,925, filed May 18, 2007, the completedisclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a method of determining the overall oxidativestatus of a body fluid or a tissue of a patient by measuring theoxidation-reduction potential (ORP) of the body fluid or tissue. Inparticular, the invention relates to methods for the diagnosis,evaluation and monitoring of patients who have suffered a trauma (suchas a head injury), patients suspected of being critically-ill or who arecritically ill, patients who have a viral infection, and patientssuspected of having a myocardial infarction (MI) or who have an MI. Theinvention also relates to methods for the evaluation and monitoring ofthe exercise performance of patients. The invention further relates tomethods for the evaluation and monitoring of stored blood products andof patients who will receive such products.

BACKGROUND

Oxidative stress is caused by a higher production of reactive oxygen andreactive nitrogen species or a decrease in endogenous protectiveantioxidative capacity. Oxidative stress has been related to variousdiseases and aging, and it has been found to occur in all types ofcritical illnesses. See, e.g., Veglia et al., Biomarkers, 11(6): 562-573(2006); Roth et al., Current Opinion in Clinical Nutrition and MetabolicCare, 7:161-168 (2004); U.S. Pat. No. 5,290,519 and U.S. PatentPublication No. 2005/0142613. Several investigations have shown a closeassociation between the oxidative status of a critically ill patient andthe patient's outcome. See Roth et al., Current Opinion in ClinicalNutrition and Metabolic Care, 7:161-168 (2004).

Oxidative stress in patients has been evaluated by measuring variousindividual markers. See, e.g., Veglia et al., Biomarkers, 11(6): 562-573(2006); Roth et al., Current Opinion in Clinical Nutrition and MetabolicCare, 7:161-168 (2004); U.S. Pat. No. 5,290,519 and U.S. PatentPublication No. 2005/0142613. However, such measurements are oftenunreliable and provide conflicting and variable measurements of theoxidative status of a patient. See Veglia et al., Biomarkers, 11(6):562-573 (2006); Roth et al., Current Opinion in Clinical Nutrition andMetabolic Care, 7:161-168 (2004). The measurement of multiple markerswhich are then used to provide a score or other assessment of theoverall oxidative status of a patient has been developed to overcome theproblems of using measurements of single markers. See Veglia et al.,Biomarkers, 11(6): 562-573 (2006); Roth et al., Current Opinion inClinical Nutrition and Metabolic Care, 7:161-168 (2004). Although suchapproaches are more reliable and sensitive than measurements of a singlemarker, they are complex and time consuming. Thus, there is a need for asimpler and faster method for reliably measuring the overall oxidativestatus of a patient.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a method of diagnosing,evaluating or monitoring a patient who has suffered a trauma comprisingthe following steps:

-   -   measuring the oxidation-reduction potential (ORP) of a body        fluid of the patient, a tissue of the patient or both; and    -   performing one or both of the following steps:    -   determining if the ORP is significantly different than the ORP        of the same body fluid, tissue or both from normal patients;        and/or    -   if the ORP of the body fluid, tissue or both of the patient has        been measured at least one time prior to the current        measurement, determining if the ORP has increased or decreased        compared to the prior measurement(s).

In some embodiments, the ORP is monitored to determine the efficacy of atreatment of the patient.

In some embodiments, the ORP is monitored to determine whether thecondition of the patient is improving or worsening.

In some embodiments, the ORP is measured once per day or a plurality oftimes per day.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is plasma.

In some embodiments, the trauma is a head injury.

In some embodiments, the ORP is measured once per day or a plurality oftimes per day.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is plasma.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is cerebrospinal fluid.

Another aspect of the invention relates to a method of diagnosing,evaluating or monitoring a patient who has a viral infection comprisingthe following steps:

-   -   measuring the oxidation-reduction potential (ORP) of a body        fluid of the patient, a tissue of the patient or both; and    -   performing one or both of the following steps:    -   determining if the ORP is significantly different than the ORP        of the same body fluid, tissue or both from normal patients;        and/or    -   if the ORP of the body fluid, tissue or both of the patient has        been measured at least one time prior to the current        measurement, determining if the ORP has increased or decreased        compared to the prior measurement(s).

In some embodiments, the ORP is monitored to determine the efficacy of atreatment of the patient.

In some embodiments, the ORP is monitored to determine whether thecondition of the patient is improving or worsening.

In some embodiments, the ORP is measured once per day or a plurality oftimes per day.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is plasma.

Another aspect of the invention relates to a method of diagnosing,evaluating or monitoring a patient suspected of being critically-ill orof evaluating or monitoring a patient who has been diagnosed as beingcritically ill, the method comprising the following steps:

-   -   measuring the oxidation-reduction potential (ORP) of a body        fluid of the patient, a tissue of the patient or both; and    -   performing one or both of the following steps:    -   determining if the ORP is significantly different than the ORP        of the same body fluid, tissue or both from normal patients;        and/or    -   if the ORP of the body fluid, tissue or both of the patient has        been measured at least one time prior to the current        measurement, determining if the ORP has increased or decreased        compared to the prior measurement(s).

In some embodiments, the ORP is used as part of an evaluation todetermine whether the patient is or is not critically ill.

In some embodiments, the ORP is monitored to determine the efficacy of atreatment of the patient.

In some embodiments, the ORP is monitored to determine whether thecondition of the patient is improving or worsening.

In some embodiments, the ORP is measured once per day or a plurality oftimes per day.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is plasma.

Another aspect of the invention relates to a method of diagnosing,evaluating or monitoring a patient suspected of having a myocardialinfarction or of evaluating or monitoring a patient who has beendiagnosed as having a myocardial infarction, the method comprising thefollowing steps:

-   -   measuring the oxidation-reduction potential (ORP) of a body        fluid of the patient, a tissue of the patient or both; and    -   performing one or both of the following steps:    -   determining if the ORP is significantly different than the ORP        of the same body fluid, tissue or both from normal patients;        and/or    -   if the ORP of the body fluid, tissue or both of the patient has        been measured at least one time prior to the current        measurement, determining if the ORP has increased or decreased        compared to the prior measurement(s).

In some embodiments, the ORP is used as part of an evaluation todetermine whether the patient is or is not having a myocardialinfarction.

In some embodiments, the ORP is monitored to determine the efficacy of atreatment of the patient.

In some embodiments, the ORP is measured once per day or a plurality oftimes per day.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is plasma.

Another aspect of the invention relates to a method of evaluating theexercise performance of a patient comprising:

-   -   measuring the oxidation-reduction potential (ORP) of a body        fluid of the patient, a tissue of the patient or both before and        after exercise and determining if the ORP is significantly        different after exercise as compared to before exercise; or    -   measuring the ORP of a body fluid of the patient, a tissue of        the patient or both before and during exercise and determining        if the ORP is significantly different during exercise as        compared to before exercise; or    -   measuring the ORP of a body fluid of the patient, a tissue of        the patient or both before, during and after exercise and        determining if the ORP is significantly different during        exercise, after exercise or both, as compared to before        exercise.

In some embodiments, the ORP is measured in a body fluid, and the bodyfluid is plasma.

Another aspect of the invention relates to a method of evaluating ormonitoring a blood product which is stored comprising:

-   -   measuring the oxidation-reduction potential (ORP) of the blood        product after it has been stored for one or more days; and    -   determining if the ORP of the stored blood product is        significantly different than the ORP of the same blood product        immediately after it was obtained from a donor.

In some embodiments, the blood product is blood.

In some embodiments, the blood product is packed red blood cells.

In some embodiments, the ORP of the blood product is measured once perday during storage.

In some embodiments, the method further comprises:

-   -   measuring the oxidation-reduction potential (ORP) of a body        fluid of a patient who is to receive the blood product, a tissue        of the patient or both; and    -   determining if the ORP is significantly different than the ORP        level in the same body fluid, tissue or both from normal        patients.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides such a method. In particular, theinvention provides a method of determining the overall oxidative statusof a body fluid or a tissue of a patient by measuring theoxidation-reduction potential (ORP) of the body fluid or tissue. Themethod has been found to be useful in the diagnosis, evaluation andmonitoring of patients who have suffered a trauma (such as a headinjury), patients suspected of being critically-ill or who have beendiagnosed as being critically ill, patients who have a viral infection,and patients suspected of having a myocardial infarction (MI) or whohave been diagnosed as having an MI. The method has also been founduseful in monitoring and evaluating exercise performance in patients. Inaddition, the method has been found useful in monitoring and evaluatingstored blood products and patients who will receive such a product.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS OF THEINVENTION

As used herein, “patient” means a mammal, such as a dog, cat, horse, cowor human. Most preferably, the patient is a human.

Any body fluid of a patient can be used in the method of the invention.Suitable body fluids include a blood sample (e.g., whole blood, serum orplasma), urine, saliva, cerebrospinal fluid, tears, semen, vaginalsecretions, amniotic fluid and cord blood. Also, lavages, tissuehomogenates and cell lysates can be utilized and, as used herein, “bodyfluid” includes such preparations. Preferably, the body fluid is blood,plasma, serum or cerebrospinal fluid. For head injuries, the body fluidis most preferably cerebrospinal fluid or plasma. In cases other thanhead injuries, the body fluid is most preferably plasma.

Any tissue of a patient can be used in the method of the invention.Suitable tissues include skin, eye and mouth tissues and tissue frombiopsies.

As used herein, “normal,” “normal patient” or “control” means a mammalof the same species as the patient (e.g., the normal will be a humanwhen the patient is a human), and who is not suffering from any disease.Since ORP increases with age, normals should be of the same age or agerange as the patient(s) being tested.

An oxidation-reduction system, or redox system, involves the transfer ofelectrons from a reductant to an oxidant according to the followingequation:oxidant+ne⁻

reductant  (1)where ne⁻ equals the number of electrons transferred. At equilibrium,the redox potential (E), or oxidation-reduction potential (ORP), iscalculated according to the Nernst-Peters equation:E(ORP)=E _(o) −RT/nF ln [reductant]/[oxidant]  (2)where R (gas constant), T (temperature in degrees Kelvin) and F (Faradayconstant) are constants. E_(o) is the standard potential of a redoxsystem measured with respect to a hydrogen electrode, which isarbitrarily assigned an E_(o) of 0 volts, and n is the number ofelectrons transferred. Therefore, ORP is dependent on the totalconcentrations of reductants and oxidants, and ORP is an integratedmeasure of the balance between total oxidants and reductants in aparticular system. As such, ORP provides a measure of the overalloxidative status of a body fluid or tissue of a patient.

An ORP measurement which is significantly higher than that of normalswill indicate the presence of oxidative stress. Oxidative stress hasbeen related to many diseases, and it has been found to occur in alltypes of critical illnesses. Accordingly, an ORP level significantlyhigher than that of normals indicates the presence of a disease andperhaps a critical illness. An ORP measurement which is the same as orlower than that of normals indicates the absence of oxidative stress andthe absence of a disease or critical illness. Thus, the ORP level of apatient can be used by a medical doctor or veterinarian as an aid indiagnosing or ruling out the presence of a disease, particularly aserious illness. Sequential measurements of ORP over time can be used tomonitor the progression of a disease and the effectiveness or lack ofeffectiveness of treatment of the disease. If a patient's ORP does notdecrease after treatment, or especially if it increases despitetreatment, this may indicate a poor prognosis and the need for moreaggressive and/or additional and/or different treatments. In the case ofa measurement made by a patient, such as a patient experiencing symptomsof myocardial infarction, the ORP level may indicate the need for thepatient to see a doctor or to immediately proceed to an emergency roomfor treatment.

The ORP of a body fluid or a tissue can be easily measure by contactingan ORP or redox electrode with the body fluid or tissue. Such electrodesare available commercially from, e.g., Microelectrodes, Inc., Bedford,N.H. Such electrodes can suffer from day-to-day variability, and the useof references standards will be necessary. Suitable references standardsinclude saturated quinhydrone at several pH's. The electrode isconnected to a meter which provides a readout of the ORP in millivoltsand, optionally, other parameters, such as pH and temperature. Suchmeters are available commercially from, e.g., Hanna Instruments,Woonsocket, R.I.

The method of the invention has been found to be useful in thediagnosis, evaluation and monitoring of patients who are suffering from,or who are suspected of having, certain diseases. ORP can be used, incombination with a medical history, symptoms and other test results, asan aid in diagnosing, evaluating and monitoring these diseases andtreatments of them. In particular, a normal ORP may be very helpful inruling out the presence of a disease, particularly a serious illness,and in saving medical resources that might otherwise be devoted tounnecessarily treating patients. In addition, a significantly high ORP(indicating oxidative stress) may be used to identify those patients whoare in need of immediate or more aggressive treatment of their diseaseand/or treatment to reduce oxidative stress.

An ORP that is significantly high compared to normals may indicate theneed or desirability of performing tests for one or more individualmarkers of oxidative stress to better understand the reason or sourceof, and therefore the best treatment for, the oxidative stress ordisease. Thus, the invention also includes the use of ORP in combinationwith tests for one or more individual markers of oxidative stress(collectively referred to herein as an “oxidative stress panel oftests”). Such markers of oxidative stress and methods of measuring themare known. See, e.g., Veglia, et al., Biomarkers, 11(6):562-573 (2006);Rana et al., Mol. Cell Biochem., 291:161-166 (2006); Roth et al., Curr.Opin. Clin. Nutr. Metab. Care, 7:161-168 (2004); Horton, Toxicology,189:75-88 (2003); Winterbourn et al., Crit. Care Med., 28:143-149(2000); Ghiselli et al., Free Radic. Biol. Med., 29(11):1106-1114(2000); Rice-Evans, Free Radic. Res., 33 Suppl.:S59-S66 (2000); Priorand Cao, Free Radic. Biol. Med., 27(11-12):1173-1181 (1999); Galley etal., Crit. Care Med., 24:1649-53 (1996); Goode et al., Crit. Care Med.,23:646-651 (1995), the complete disclosures of which are incorporatedherein by reference.

Trauma injury is a leading cause of death and disability for all agegroups under 60 years of age. In the United States, trauma injuriesaccount for more than 160,000 deaths each year and millions moresurvivable injuries. For many people, the injury causes temporary painand inconvenience. For others, the injury leads to suffering,disability, chronic pain, and a profound change in quality of life,including substantial financial consequences. The economic costs oftrauma injuries include the costs associated with medical treatment aswell as lost productivity costs. In 2000 alone, the 50 million injuriesthat required medical treatment will ultimately cost $406 billion. Thisincludes estimates of $80.2 billion in medical care costs and $326billion in productivity losses.

The method of the present invention has been found to be useful in thediagnosis, evaluation and monitoring of patients who have suffered atrauma. As used herein, “trauma” refers to physical injury to any partof a patient's body or to multiple parts of a patient's body. Traumainjuries include head injuries, internal injuries, blunt trauma,multiple trauma, broken bones and burns.

The present invention provides a means for the determination andmonitoring of the oxidative status of traumatized patients and providesmedical doctors and veterinarians with real-time information to guidetrauma treatment and care. In particular, the existence and degree ofoxidative stress in traumatized patients can be determined andmonitored. For example, an ORP level that is statistically the same as,or lower than, that of normals upon first examination of a traumapatient (e.g., by a paramedic in the field or by a medical doctor in anemergency room) indicates that oxidative stress is not present and thatthe patient may not need aggressive treatment or may not even need to beadmitted to a hospital. In this manner, medical resources can be savedand costs lowered. On the other hand, an ORP level significantly higherthan that of normals upon first examination of a trauma patientindicates the presence of oxidative stress and the need for immediatetreatment of the patient and for continued monitoring of the patient'sORP. The higher the ORP level, the higher the level of oxidative stress,and the greater the need for aggressive treatment of the patient. An ORPlevel that decreases with treatment indicates that the patient isimproving and that the treatment is working. An ORP level that increasesdespite treatment indicates that the patient is getting worse and thatmore aggressive treatment, additional treatment and/or a differenttreatment is needed. An ORP level that has decreased to the point whereit is no longer significantly higher than that of normals indicates thatthe patient may be discharged from the hospital. Of course, the ORPlevel is only one diagnostic parameter, and it should be used incombination with other symptoms, results of a physical examination, amedical history, and the results of any other laboratory tests, todetermine the proper treatment for a trauma patient.

The method of the invention has also been found to be useful in thediagnosis, evaluation and monitoring of patients who have a viralinfection. Viral infections include infections caused by humanimmunodeficiency virus, encephalitis virus, hepatitis viruses, influenzaviruses, pneumonia virus and other viruses that cause serious viralillnesses.

In a patient suffering from a viral infection, an ORP level that isstatistically the same as, or lower than, that of normals uponexamination of such a patient indicates that oxidative stress is notpresent and that the patient may need only standard treatments. On theother hand, an ORP level significantly higher than that of normals uponexamination of a patient indicates the presence of oxidative stress andthe need for more aggressive treatment of the patient, includingpossibly hospital admission, and for continued monitoring of thepatient's ORP. The higher the ORP level, the higher the level ofoxidative stress, and the greater the need for aggressive treatment ofthe patient. An ORP level that decreases with treatment indicates thatthe patient is improving and that the treatment is working. An ORP levelthat increases despite treatment indicates that the patient is gettingworse and that more aggressive treatment, additional treatment and/or adifferent treatment is needed. An ORP level that has decreased to thepoint where it is no longer significantly higher than that of normalsindicates that aggressive treatment of the patient may be discontinued,including discharge of the patient from the hospital. Of course, the ORPlevel is only one diagnostic parameter, and it should be used incombination with other symptoms, results of a physical examination, amedical history, and the results of any other laboratory tests, todetermine the proper treatment for a patient having a viral infection.

Each year in the United States, approximately 6-8 million people presentto a hospital emergency room (ER) with chest pain or other cardiacsymptoms (e.g., shortness of breath and pain or tingling in the leftarm). Unfortunately, about 2-5% of the 3-4 million that are sent homefrom the ER are mistakenly diagnosed. Chest pain diagnostic errors arethe leading cause of emergency medicine malpractice awards. Of the other3-4 million that are hospitalized, about 60-75% do not have cardiacdisease. The minimum cost for each hospitalized patient is $3,000-5,000,which means that over 6 billion healthcare dollars are wasted each yearbecause of these unnecessary hospitalizations. With a non-diagnosticelectrocardiogram (ECG), reliable early biomarkers do not exist.Troponin I or troponin T levels are unreliable during the first 6-24hours after the onset of symptoms due to low sensitivity, and creatinekinase isoenzymes (CK-MB) and myoglobin are not cardiac specific. Itwould be highly desirable to have a laboratory test result that couldaid in the diagnosis of myocardial infarction or rule it out.

The method of the invention provides such a test result and has beenfound to be useful in the diagnosis, evaluation and monitoring ofpatients suspected of having a myocardial infarction (MI). The method ofthe present invention is particularly useful for the early diagnosis ofMI. By “early diagnosis” is meant ascertaining the presence or absenceof MI during the first few hours (less than 24 hours, especially lessthan 12 hours) following the onset of symptoms indicative of MI, such aschest pain, shortness of breath and pain or tingling in the left arm.The method of the invention also has been found to be useful in theevaluation and monitoring of patients who have been diagnosed with anMI.

In particular, the existence and degree of oxidative stress in patientspresenting with symptoms of MI can be determined and monitored accordingto the present invention. For example, an ORP level that isstatistically the same as, or lower than, that of normals upon firstexamination of a suspected MI patient (e.g., by a paramedic in the fieldor by a medical doctor in an emergency room) indicates that oxidativestress is not present and that the patient is not experiencing an MI. Insuch a case, the patient may not need treatment and may not need to bekept in an ER or admitted to the hospital. In this manner, medicalresources can be saved and costs lowered. On the other hand, an ORPlevel significantly higher than that of normals upon first examinationof a suspected MI patient indicates the presence of oxidative stress andthat the patient may experiencing an MI. Such an ORP level indicates theneed for immediate treatment of the patient and continued monitoring ofthe patient's ORP. The higher the ORP level, the higher the level ofoxidative stress, and the greater the need for aggressive treatment ofthe patient. An ORP level that decreases with treatment indicates thatthe patient is improving and that the treatment is working. An ORP levelthat increases despite treatment indicates that the patient is gettingworse and that more aggressive treatment, additional treatment and/or adifferent treatment is needed. An ORP level that has decreased to thepoint where it is no longer significantly higher than that of normalsindicates that the patient may be discharged from the hospital. Ofcourse, the ORP level is only one diagnostic parameter, and it should beused in combination with other symptoms, results of a physicalexamination, a medical history, and the results of any other laboratorytests, to determine the proper treatment for a suspected MI patient or apatient diagnosed as actually experiencing an MI.

The method of the invention has also been found to be useful in thediagnosis, evaluation and monitoring of patients suspected of beingcritically ill and evaluation and monitoring of patients who are foundto be critically ill. It is well known that the presence of oxidativestress in critically ill patients is positively correlated with pooroutcomes. See Roth et al., Curr. Opin. Clin. Nutr. Metab. Care,7:161-168 (2004). Accordingly, the ORP of patients who are, who aresuspected of being, or who are likely to become, critically-ill shouldbe monitored. An ORP level of a patient that is statistically the sameas, or lower than, that of normals indicates that oxidative stress isnot present and that the patient is not critically ill. Such an ORPlevel indicates that aggressive treatment of the patient is not needed.An ORP level that is significantly higher than that of normals indicatesthe presence of oxidative stress and that the patient is critically ill.Such an ORP level indicates the need for aggressive treatment of thepatient and for continued monitoring of the patient's ORP. The higherthe ORP level, the higher the level of oxidative stress, and the greaterthe need for aggressive treatment of the patient. An ORP level thatdecreases with treatment indicates that the patient is improving andthat the treatment is working. An ORP level that increases despitetreatment indicates that the patient is getting worse and that moreaggressive treatment, additional treatment and/or a different treatmentis needed. An ORP level that has decreased to the point where it is nolonger significantly higher than that of normals indicates that thepatient is no longer critically ill and may be discharged from thehospital. Of course, the ORP level is only one diagnostic parameter, andit should be used in combination with other symptoms, results of aphysical examination, a medical history, and the results of any otherlaboratory tests, to determine the proper treatment for a patient.

The use of banked blood products is a common practice employed by themedical community worldwide for obvious beneficial reasons. However,there is a risk of adverse side effects from the transfusion of bloodproducts into patients, including the possible development oftransfusion-related acute lung injury (TRALI), multiple organ failure(MOF), and acute respiratory distress syndrome (ARDS). Brittingham etal., J. Am. Med. Assoc., 165:819-825 (1957); Sauaia et al., Arch. Surg.,129:39-45 (1994); Miller et al., Am. Surg., 68:845-850 (2002). It wouldbe desirable to have a means of avoiding or reducing these side effects.

The method of the invention provides such a means, and the method of thepresent invention has been found useful in monitoring and evaluatingstored (banked) blood products. Blood products that can be monitored andevaluated according to the present invention include whole blood, packedred blood cells, platelets and fresh frozen plasma. In particular, usingthe method of the present invention, it has been found the ORP of storedblood products increases with the time of storage. For example, packedred blood cells have a significantly increased ORP on day 42 as comparedto day 1. An increased ORP indicates an increased level of oxidants inthe blood product, and the increased level of oxidants may contribute tothe development of side effects in patients receiving the blood product,since transfusion of such a blood product would be expected to increasethe level of oxidants and oxidative stress in the patient.

The method of the invention is also useful in monitoring and evaluatingpatients who will receive stored blood products. In particular, an ORPlevel in such a patient which is significantly higher than that ofnormals indicates the presence of oxidative stress, and such a patientshould not be transfused or should be transfused with blood productsthat contain lower levels of oxidant species (i.e., a blood product thathas a lower ORP level, preferably the same as that of fresh bloodproduct). The ORP level of a blood product may be less critical forpatients who have an ORP level that is statistically the same as, orlower than, that of normals, since such a level indicates that oxidativestress is not present. Determining the oxidative status of the patientand of the blood product should result in a decrease intransfusion-related side effects.

Exercise is associated with an enhanced aerobic and/or anaerobicmetabolism which results in an increased formation of reactive oxygenspecies (ROS). Strenuous exercise, excessive exercise and overtraininggenerate ROS to a level that can overwhelm antioxidant defense systems.See Sen, Sports Med., 31:891-908 (2001); Margonis et al., Free RadicalBiol. Med., 43(6):901-910 (Sep. 15, 2007); Gomez-Cabrera et al., FreeRadical Biol. Med., 44(2):126-131 (2008); Radak et al., Ageing Res.Rev., 7(1):34-42 (2008). The result is oxidative stress, and oxidativestress can cause extensive molecular, cellular and tissue damage. Onepossible outcome is oxidative damage to muscle tissues. Preventing orreducing muscle tissue damage during exercise training should helpoptimize the training effect and eventual performance. Regular exerciseof moderate intensity and duration, while generating ROS, also inducesan improvement in natural antioxidant enzymes and proteins andupregulation of antioxidant defense systems. See Ji, Proc. Soc. Exp.Biol. Med., 222:283-292 (1999); Rahnama et al., J. Sports Med. Phys.Fitness, 47:119-123 (2007); Gomez-Cabrera et al., Free Radical Biol.Med., 44(2):126-131 (2008); Ji, Free Radical Biol. Med., 44:142-152(2008); Radak et al., Ageing Res. Rev., 7(1):34-42 (2008). Theseadaptations result in decreased oxidative challenge to the body andmaintenance of the oxidant-antioxidant homeostasis. Further, it appearsthat exercise-induced modulation of the redox state is an importantmeans by which exercise benefits the body, increasing the resistanceagainst, and facilitating recovery from, oxidative stress. Radak et al.,Appl. Physiol. Nutr. Metab., 32:942-946 (2007); Radak et al., FreeRadical Bil. Med., 44:153-159 (2008). From the foregoing, it can be seenthat the maximum benefits of exercise can be realized from exercise thatdoes not cause oxidative stress, and that exercise that does causeoxidative stress is to be avoided, whenever possible.

The method of the invention can be used to monitor and evaluate exerciseperformance in patients. The ORP of patients before and after, beforeand during, or before, during and after exercise is measured. An ORPlevel of a patient during or after exercise that is statistically thesame as, or lower than, that of the patient before exercise indicatesthat oxidative stress is not present. Such an ORP level indicates thatthe exercise need not be changed. An ORP level of a patient during orafter exercise that is significantly higher than that of the patientbefore exercise indicates the presence of oxidative stress. Such an ORPlevel indicates that the exercise may be causing damage and should bechanged in some way, such as changing the frequency of the exercise,length of the exercise or even the type of exercise. As can be seen,monitoring the ORP of a patient can result in the design of an optimumexercise program for the patient so that the patient can achieve his/herdesired physical fitness goals without experiencing the adverse effectsand damage caused by oxidative stress.

EXAMPLES Example 1 Diagnosis of Acute Myocardial Infarction

Whole blood was collected from normal subjects and patients with acutemyocardial infarction (AMI) by venipuncture using a VACUTAINER™containing sodium heparin (Becton Dickinson, Franklin Lakes, N.J., USA).Plasma was aliquoted in 1 mL quantities and stored at −80° C. for futureuse.

Oxidation-reduction potential (ORP) measurements were recorded using amicro Pt/AgCl combination MI-800/410 cm Redox Electrode(Microelectrodes, Inc., Bedford, N.H., USA) connected to an HI4222pH/mV/Temperature bench meter (Hanna Instruments, Woonsocket, R.I.,USA). The electrode was immersed in a plasma sample, and a steady-stateORP reading in millivolts (mV) was recorded.

The results are presented in Table 1 below. The data were analyzed usingstudent's two-tailed t test assuming uneven variances. As can be seenfrom Table 1, the ORP of the plasmas from AMI patients was significantlydifferent than that of the plasmas from normals. Thus, a plasma ORPmeasurement can be used as an aid in distinguishing patients who areactually experiencing an AMI from those whose AMI-like symptoms are dueto another cause.

TABLE 1 Sample # ORP AMI Plasmas GR-358 −24.5 GR-379 −30.5 GR-397 −34.0GR-1266-05 −15.0 GR-1328 −4.1 GR-1435-02 −11.7 Average: −20.0 SD: 11.6Normal Plasmas GR-1347-02 −41.7 GR-1425 −65.3 GR-1426 −52.6 GR-1427−52.7 GR-1428 −59.2 GR-1429 −55.3 GR-1430 −53.2 GR-1431 −58.4 GR-1432−30.6 GR-1433 −50.9 Average: −52.0 SD: 9.7 Comparison AMI samples withnormal samples p-value 0.0003

Example 2 Monitoring and Identifying Critically Ill Trauma Patients

The oxidation-reduction potential (ORP) in a biological system is anintegrated measure of the balance between total pro- and antioxidantcomponents of the system. In plasma, many constituents contribute to theORP. Reactive oxygen species (ROS), such as the superoxide ion, hydroxylradical, hydrogen peroxide, nitric oxide, peroxynitrite, transitionmetal ions, and hypochlorous acid, contribute to the oxidativepotential. Plasma antioxidants include thiols, vitamin C, tocopherol,β-carotene, lycopene, uric acid, bilirubin, and flavinoids. Enzymes,such as SOD, catalase, and glutathione peroxidase, are involved in theconversion of ROS into less reactive species. ORP monitoring of plasmaprovides a single measurement that integrates the overall quantitativebalance among the pro- and antioxidant components of the system, and theORP level is an indicator of a patient's overall oxidative status.

Critically ill patients suffer from oxidative stress, reflecting animbalance in favor of the pro-oxidant components in the intra- andextracellular environments. The biological consequences of thisimbalance favor certain chemical reactions which could be bothbeneficial and detrimental to the system depending upon the systeminvolved and the disease process. Previous attempts at assessing theredox status of critically ill patients have been limited tomeasurements of single parameters, such as concentrations of individualantioxidants (Goode et al., Crit. Care Med., 23:646-51 (1995)) or amountof lipid peroxidation (Rana et al., Mol. Cell Biochem., 291:161-6(2006)). Although these parameters could be helpful, they might not givethe clinician a complete assessment of the amount of oxidative stressoccurring in a critically ill patient. Additionally, the measurement ofthese various parameters would prove to be laborious, time consuming,and, hence, impractical in the clinical setting. Here, a method isdescribed that measures the overall oxidative status of critically illtrauma patients using an electrode that measures ORP in the plasma ofthe patients on a possible real-time basis.

Materials and Methods

This study received approval by the HCA-HealthOne Institutional ReviewBoard according to the guidelines published by the HHS Office forProtection from Research Risk. Blood was collected from normal subjects(N=10) and critically ill patients who had suffered severe traumas(N=39) by venipuncture using a VACUTAINER™ containing sodium heparin(Becton Dickinson, Franklin Lakes, N.J., USA). For critically illpatients, blood was collected on an almost daily basis until discharge.Plasma was aliquoted in 1 mL quantities and stored at −80° C. for futureuse. Patient demographics are listed in Table 2.

TABLE 2 Patients Controls Number (n) 39 10 Age 43.8 yrs ± 2.7 SEM 46.4yrs ± 3.5 SEM Sex 31 males 7 males 8 females 3 females Injury SeverityScore (ISS) 30.7 ± 2.4 SEM N/A

Oxidation-reduction potential (ORP) measurements were recorded at roomtemperature using a micro Pt/AgCl combination MI-800/410 cm RedoxElectrode (Microelectrodes, Inc., Bedford, N.H., USA) connected to anHI4222 pH/mV/Temperature bench meter (Hanna Instruments, Woonsocket,R.I., USA). Plasma samples were thawed, and the ORP electrode wasimmersed in the plasma. A reading was recorded in millivolts (mV) afterthe ORP value was stable for 5 seconds.

Total protein was quantified in all plasma samples using thebicinchoninic acid protein assay (Pierce Biotechnology, Rockford, Ill.,USA). All plasma samples were diluted 1:100 with 1× phosphate-bufferedsaline, pH 7.4, prior to application to a 96-well plate. All sampleswere analyzed in duplicates.

Paraoxonase (PON) is a calcium-dependent aryl esterase (AE) present inplasma. When PON is associated with high density lipoproteins (HDL), anantioxidant effect against lipid peroxidation has been observed(Ferretti et al., Biochim. Biophys. Acta, 1635:48-54 (2003)). Indeed, alower PON activity has been associated with a higher susceptibility ofHDL to peroxidation in patients affected by diseases characterized byincreases in oxidative damage (Ferretti et al., J. Clin. Endocrinol.Metab., 89:2957-2962 (2004); Ferretti et al., J. Clin. Endocrinol.Metab., 90:1728-1733 (2005)).

Plasma PON-AE activity was measured as previously described. Ahmed etal., J. Biol. Chem., 280:5724-5732 (2005). Briefly, plasma was diluted1:20 with 1× phosphate-buffered saline, pH 7.4. Diluted plasma was thencombined 1:1 with 4 mM para-nitrophenolacetate (Sigma-Aldrich, St.Louis, Mo.) in a 96-well plate in duplicates. The plate was immediatelyread on a pre-warmed (37° C.) plate reader (FL600 MicroplateFluorescence Reader, Bio-Tek Instruments Inc., Winooski, Vt.) set at 410nm. Absorbance readings were taken every 2 minutes for 20 minutes. Theslope of the linear portion of the kinetic plot (R²≧0.99) was used togenerate PON-AE activity. PON-AE activity was normalized to plasmaprotein. PON-AE activity is reported in units (U), where a unit equalsthe change in milli-absorbance units at 410 nm per minute per mg oftotal plasma protein.

The ORP, PON-AE and total protein data were analyzed using Matlab R14(Mathworks, Natick, Mass.). A one-way ANOVA was used to compare allpatient data versus normal data to test for significant differences witha Tukey-Kramer correction for multiple comparisons with a significancelevel of 0.05. All data are reported as ±standard error of the mean(SEM).

Results and Conclusions

Plasma was collected from critically ill patients who had sufferedsevere traumas from the time of admission until discharge. ORP wasmeasured in the complete series of plasma samples collected from aparticular patient after the patient was discharged in order to limitany day-to-day variability in the ORP electrode.

A rapid increase in ORP was observed for all of the patients after aninitial ORP reading of −19.9 mV (±3.0 SEM) on admission. The admissionORP value is significantly higher than that of normal plasmas (−52.0mV±3.1 SEM, p<0.05). The ORP maximum, reached at day 6 (±0.5 SEM), wassignificantly higher than the admission value, with a value of +13.7 mV(±2.5 SEM). The ORP maximum was also significantly higher than normalplasmas (−52.0 mV±3.1 SEM, p<0.05). During the remaining course of thehospitalization, the ORP of the plasma of these severely traumatizedpatients steadily approached the ORP of normal plasma. At the time ofdischarge, the ORP of plasma obtained from the severely traumatizedpatients was not significantly different from that of normal plasma(−52.0 mV±3.1 SEM).

For method validation purposes, PON-AE activities and total proteinlevels were measured. PON-AE activities were significantly higher in theadmission samples (740.0 U/mg protein±20.2 SEM) compared to the ORPmaxima samples (649.1 U/mg protein±18.8 SEM). Thus, the results show acorrelation between trauma and a decrease in PON-AE activity. Similarly,protein levels were higher in the admission samples (47.3 mg/ml±1.6 SEM)compared to the ORP maxima samples (41.6±1.3 SEM).

The presence of markers of oxidative stress in critically ill patientsis associated with a poor prognosis (Roth et al., Curr. Opin. Clin.Nutr. Metab. Care, 7:161-8 (2004)). However, no single parameter canaccurately predict the overall redox status for a critically illpatient. The laborious performance of multiple assays for thequantification of pro-oxidants and antioxidants is not practical in aclinical setting. Therefore, a quick and simple diagnostic test iswarranted.

Here, the oxidation-reduction potential (ORP) of plasma collected onconsecutive days from critically ill, traumatized patients was measuredfrom hospital admission until the time of discharge. Daily ORP levelscorrelated with clinical events recorded in the medical records of eachparticular patient, with increases in ORP indicating a worsening of apatient's condition and decreases in ORP indicating an improvement in apatient's condition. Accordingly, monitoring ORP should be a useful toolfor assessing and monitoring the presence and degree of oxidativestress, the severity of injury, a patient's prognosis, and the efficacyof treatment(s). ORP monitoring could be used to determine theappropriate clinical conditions and timing that warrant treatment (e.g.,administration of antioxidants) of trauma patients. In particular, ORPmonitoring could be used to aid in the identification of patients whoare critically ill and those who need more aggressive treatment than maybe indicated by their symptoms and other diagnostic test results.

Example 3 ORP of Plasmas Before and after Exercise

The purpose of this experiment was to determine if exercise had aneffect on oxidation-reduction potential (ORP) in plasma. Whole blood wascollected from members of a women's college soccer team before and afterexercise. The exercise consisted of an intense one-hour cardiovascularworkout. The blood was collected by venipuncture using a VACUTAINER™containing sodium heparin (Becton Dickinson, Franklin Lakes, N.J., USA).Blood tubes were centrifuged at 2000 rpm for 10 minutes, and plasma wascollected and aliquoted in 1 mL quantities and stored at −80° C. forfuture use.

The plasma samples were thawed at room temperature. ORP measurementswere recorded using an MI-800/410 cm Redox Electrode (Microelectrodes,Inc., Bedford, N.H., USA) connected to an HI4222 pH/mV/Temperature benchmeter (Hanna Instruments, Woonsocket, R.I., USA). The electrode wasimmersed in a plasma sample, and a steady-state ORP reading inmillivolts (mV) was recorded.

The results are presented in Table 3 below. As can be seen from Table 3,the ORP of the plasmas of these high performance athletes decreasedafter exercise.

TABLE 3 Patient Sample Exercise ORP (mV) Change (+/−) GR 1814-01 No−39.4 GR 1814-02 Yes −46.0 −6.6 GR 1815-01 No −35.5 GR 1815-02 Yes −38.3−2.8 GR 1816-01 No −40.7 GR 1816-02 Yes −44.4 −3.7 GR 1817-01 No −49.5GR 1817-02 Yes −49.2 +0.3 Average change −3.2

Example 4 ORP of Plasma from Critically-Ill Patient Suffering ViralInfection

Patient GR-1029 was admitted to the intensive care unit of SwedishHospital, Englewood, Colo. with flu-like symptoms, pneumonia andrespiratory failure triggered by exposure to rat droppings and urinewhile cleaning the cage of a pet rat. The patient was diagnosed aslikely having a viral infection transmitted by rodents.

This study received approval by the HCA-HealthOne Institutional ReviewBoard according to the guidelines published by the HHS Office forProtection from Research Risk.

Blood was collected from this patient on several days during hishospitalization (see Table 4 below) by venipuncture using a VACUTAINER™containing sodium heparin (Becton Dickinson, Franklin Lakes, N.J., USA).Plasma was aliquoted in 1 mL quantities and stored at −80° C. for futureuse.

The plasma samples were thawed at room temperature. ORP measurementswere recorded using an MI-800/410 cm Redox Electrode (Microelectrodes,Inc., Bedford, N.H., USA) connected to an HI4222 pH/mV/Temperature benchmeter (Hanna Instruments, Woonsocket, R.I., USA). The electrode wasimmersed in a plasma sample, and a steady-state ORP reading inmillivolts (mV) was recorded.

The results are presented in Table 4 below. As can be seen from Table 4,ORP was highest during the first 8 days after admission and thendeclined until the patient was discharged, in a similar manner tocritically ill trauma patients (see Example 2). However, the ORP levelfor patient GR-1029 did not return to normal levels (−52.0 mV±3.1 SEM)before discharge. The ORP levels correlated with clinical eventsrecorded in the medical records of the patient, with increased ORPlevels indicating a worsening of the patient's condition and decreasedORP levels indicating an improvement in the patient's condition.

TABLE 4 TIME (days) ORP (mV) 1.0 73.0 1.5 78.8 8.0 59.5 9.0 36.6 10.060.6 11.0 17.7 12.0 33.6 16.0 3.8 22.0 −13.2 23.0 −12.4 24.0 −26.3 25.0−14.0 26.0 1.7 30.0 −34.2 31.0 −20.9 33.0 −7.8 36.0 −9.5 37.0 −15.8 38.0−20.0

Example 5 Monitoring of Stored Blood Product (PBRC)

Transfusion-related acute lung injury (TRALI) is an adverse effect oftransfusion and is the leading cause of transfusion-related death.Silliman et al., Blood, 105:2266-2273 (2005). Longer storage times ofpacked red blood cells (PBRCs) and other blood products have beenassociated with an increased risk in developing TRALI in transfusedpatients. See Biffl et al., J. Trauma, 50:426-432 (2001).

A total of 10 transfusion bags containing PBRCs stored in adenine,citrate and dextrose (ACD) buffer at 4° C. according to AmericanAssociation of Blood Banks criteria were obtained from Bonfils BloodCenter (Denver, Colo.). At Bonfils, a sample of each bag of PBRCs wascollected on storage days 1 and 42. Samples were immediately centrifugedat 1000 g at 4° C. for 10 minutes, and the supernatants were collectedand stored at −80° C. until further analysis.

Oxidation-reduction potential (ORP) was measured at room temperature inboth the day 1 and day 42 sample supernatants. ORP measurements wererecorded using a micro Pt/AgCl MI-800/410 cm Redox Electrode(Microelectrodes, Inc., Bedford, N.H., USA) connected to an HI4222pH/mV/Temperature bench meter (Hanna Instruments, Woonsocket, R.I.,USA). The electrode was immersed in a sample supernatant, and asteady-state ORP reading in millivolts (mV) was recorded.

The results are presented in Table 5 below. A student t-test was used tocompare day 1 versus day 42 data to test for significant differences(p<0.05, Microsoft Excel). As can be seen, ORP was significantlyincreased (p<0.05) in the day 42 samples (98.1 mV±21.9 SD) as comparedto the day 1 samples (62.6 mV±21.5 SD).

For method validation purposes, protein oxidation in the samplesupernatants was determined by measuring plasma proteins in thesupernatants by mass spectrometry (MS). Sample supernatants wereanalyzed by HPLC (Waters 2795 Separations Module, Milford, Mass., USA)coupled to positive electrospray ionization time of flight massspectrometry (+ESI-TOF MS, LCT, Micromass, UK) using a method describedpreviously. Bar-Or et al., Crit. Care Med., 33:1638-1641 (2005). 10 μLof each sample was injected onto a YMC-Pack Protein-RP HPLC column(Waters, Milford, Mass., USA) heated to 50° C. A 20-minute lineargradient from 10 to 40% B using water/0.1% trifluoroacetic acid (A) andAcN/0.1% TFA (B) was utilized with a flow rate of 1 mL/min. For eachplasma protein detected, the MS spectrum was deconvolved to theuncharged, parent mass using MaxEnt 1 software (Micromass, UK). Theparent mass spectrum was then integrated and relative proportions ofeach species were calculated using an advanced, proprietary MSintegration software package developed in-house.

Oxidation modifications of human serum albumin (HSA) includecysteinylation of cysteine 34 and dehydroalanine (DHA) modification oflysine 487. The percentage of oxidized HSA species increasedsignificantly in the supernatants from day 1 (44.1%±6.9 SD) to day 42(72.1%±8.4 SD).

Other plasma proteins identified in the supernatants by MS were α-chainof hemoglobin (αHb), β-chain of hemoglobin (βHb), apolipoprotein A1(ApoA1) and transthyretin (TTR). Significantly higher levels ofoxidation modifications of αHb, βHb and TTR were observed in the day 42supernatants as compared to the day 1 supernatants (p<0.05). Also, forαHb and ApoA1, species which had a cleaved C-terminal amino acid wereobserved, indicating the presence of carboxypeptidase activity, a markerof inflammation.

The data demonstrate the presence of an oxidative environment in PBRCs,which increases with storage time. This could partially explain theincreased risk of developing TRALI related to the transfusion of olderblood products.

Accordingly, the ORP of PBRCs and other stored blood products should bemonitored, and the ORP of patients that are to receive the bloodproducts should also be monitored. A patient that has significantoxidative stress (i.e., has a high ORP level) should be transfused withfresher blood products that contain less pro-oxidant species (i.e., hasa lower ORP level). Taking the oxidative status of the patient and ofthe blood product into account should result in a decrease intransfusion-related risk factors such as TRALI.

TABLE 5 PBRC Sample No. Day 1 ORP (mV) Day 42 ORP (mV) 054A 41.4 88.5056A 48.9 113.8 057A 105.2  124.6 058A 77.4 131.2 059A 70.3 115.5 060A88.0 100.7 061A 48.8 80.6 062A 52.2 73.5 063A 46.8 75.2 064A 46.7 77.1Average: 62.6 98.1 Standard deviation (SD) 21.5 21.9 Standard error ofthe mean  6.8 6.9 (SEM) p-value   0.0018 % Change  +56.7%

Example 6 Monitoring and Identifying Traumatic Brain Injury Patients

In this experiment, the overall oxidative status of patients withisolated, traumatic brain injuries (ITBI) was determined by measuringthe oxidation-reduction potential (ORP) of the plasma of the patients.

Serial whole blood samples were obtained from severe ITBI patients(Abbreviated Injury Score (AIS)≧3, N=32) and demographically similarnon-head injury traumatized patients (N=26) on an almost daily basisuntil discharge from the hospital. Whole blood was also collected frompatients with minor to moderate ITBI (AIS≦2, N=18) and healthyvolunteers (N=22). Plasma was aliquoted in 1 mL quantities and stored at−80° C. for future use.

ORP measurements were recorded at room temperature using a micro Pt/AgClcombination MI-800/410 cm Redox Electrode (Microelectrodes, Inc.,Bedford, N.H., USA) connected to an HI4222 pH/mV/Temperature bench meter(Hanna Instruments, Woonsocket, R.I., USA). Plasma samples were thawed,and the ORP electrode was immersed in the plasma. A reading was recordedin millivolts (mV) after the ORP value was stable for 5 seconds.

Plasma ORP increased during the first few days of hospitalization andapproached normal levels upon discharge in all trauma groups. ORP uponpresentation was significantly elevated in all traumatized patientscompared to healthy controls (p<0.05). Maximum ORP was detected on day 6for severe ITBI and non-head injury traumatized patients. However,maximum ORP values were significantly higher (p<0.05) in the severe ITBIgroup (+8.5 mV±3.4 SEM) compared to the non-head injury group (−5.2mV±2.9 SEM).

These results demonstrate the presence of an oxidative environment inthe plasma of traumatized patients, including especially severe ITBIpatients. Therefore, monitoring ORP is a useful tool for assessing thedegree of oxidative stress, severity of injury and efficacy of treatmentin ITBI patients.

We claim:
 1. A method of determining when to begin treatment of apatient having a broken bone, the method comprising: a) at the time offirst examination of the patient, measuring the oxidation-reductionpotential (ORP) of a body fluid from the patient by contacting the bodyfluid with an electrode connected to a machine that provides a read outof the ORP; and, b) comparing the ORP of the body fluid with a normalORP value; c) immediately beginning treatment of the patient if the ORPvalue of the body fluid is statistically greater than the normal ORPvalue.
 2. The method of claim 1, wherein the treatment is antioxidanttherapy.
 3. The method of claim 1 wherein the ORP is measured in plasmafrom the patient.